Abstract

The identification of enhancers with predicted specificitiesin vertebrate genomes remains a significant challenge that is hampered bya lack of experimentally validated training sets. In this study, weleveraged extreme evolutionary sequence conservation as a filter toidentify putative gene regulatory elements and characterized the in vivoenhancer activity of human-fish conserved and ultraconserved1 noncodingelements on human chromosome 16 as well as such elements from elsewherein the genome. We initially tested 165 of these extremely conservedsequences in a transgenic mouse enhancer assay and observed that 48percent (79/165) functioned reproducibly as tissue-specific enhancers ofgene expression at embryonic day 11.5. While driving expression in abroad range of anatomical structures in the embryo, the majority of the79 enhancers drove expression in various regions of the developingnervous system. Studying a set of DNA elements that specifically droveforebrain expression, we identified DNA signatures specifically enrichedin these elements and used these parameters to rank all ~;3,400human-fugu conserved noncoding elements in the human genome. The testingof the top predictions in transgenic mice resulted in a three-foldenrichment for sequences with forebrain enhancer activity. These datadramatically expand the catalogue of in vivo-characterized human geneenhancers and illustrate the future utility of such training sets for avariety of iological applications including decoding the regulatoryvocabulary ofmore » the human genome.« less

@article{osti_919760,
title = {In Vivo Enhancer Analysis Chromosome 16 Conserved NoncodingSequences},
author = {Pennacchio, Len A. and Ahituv, Nadav and Moses, Alan M. and Nobrega,Marcelo and Prabhakar, Shyam and Shoukry, Malak and Minovitsky, Simon and Visel,Axel and Dubchak, Inna and Holt, Amy and Lewis, Keith D. and Plajzer-Frick, Ingrid and Akiyama, Jennifer and De Val, Sarah and Afzal, Veena and Black, Brian L. and Couronne, Olivier and Eisen, Michael B. and Rubin, Edward M.},
abstractNote = {The identification of enhancers with predicted specificitiesin vertebrate genomes remains a significant challenge that is hampered bya lack of experimentally validated training sets. In this study, weleveraged extreme evolutionary sequence conservation as a filter toidentify putative gene regulatory elements and characterized the in vivoenhancer activity of human-fish conserved and ultraconserved1 noncodingelements on human chromosome 16 as well as such elements from elsewherein the genome. We initially tested 165 of these extremely conservedsequences in a transgenic mouse enhancer assay and observed that 48percent (79/165) functioned reproducibly as tissue-specific enhancers ofgene expression at embryonic day 11.5. While driving expression in abroad range of anatomical structures in the embryo, the majority of the79 enhancers drove expression in various regions of the developingnervous system. Studying a set of DNA elements that specifically droveforebrain expression, we identified DNA signatures specifically enrichedin these elements and used these parameters to rank all ~;3,400human-fugu conserved noncoding elements in the human genome. The testingof the top predictions in transgenic mice resulted in a three-foldenrichment for sequences with forebrain enhancer activity. These datadramatically expand the catalogue of in vivo-characterized human geneenhancers and illustrate the future utility of such training sets for avariety of iological applications including decoding the regulatoryvocabulary of the human genome.},
doi = {10.1038/nature05295},
journal = {Nature},
number = 7118,
volume = 444,
place = {United States},
year = {Wed Feb 01 00:00:00 EST 2006},
month = {Wed Feb 01 00:00:00 EST 2006}
}

Human myocyte-specific enhancer binding factor 2C (hMEF2C) belongs to the MEF2 subfamily of the MADS (MCM1, AGAMOUS, DEF A, serum response factor) family of transcription factors. Members of the MADS family share a conserved domain - the MADS domain - that is necessary for DNA binding. Highly conserved versions of the MADS domain and of an adjacent domain that is known as the MEF2 domain are found in members of the MEF2 subfamily. Both of these domains are necessary for binding to the MEF2 regulatory element. This regulatory element is known to be functionally important in a variety of muscle-specificmore » genes and possibly in the brain creatine kinase gene. The MEF2C gene product activates transcription by binding to the MEF2 element. hMEF2C is expressed at high levels in postmitotic neurons in the brain, where it is most abundant in the cerebral cortex, and is also expressed in differentiated myotubes. Several lines of evidence suggest the existence of a rat homologue of MEF2C, and a mouse homologue has been cloned. The mouse gene was mapped to mouse chromosome 13 in a region that is syntenic to human 5q13-q15. 12 refs., 1 fig.« less

Genomic sequence comparisons between human, mouse and pufferfish (Takifugu rubripes (Fugu))have revealed a set of extremely conserved noncoding sequences. While this high degree of sequence conservation suggests severe evolutionary constraint and predicts a lack of tolerance to change in order to retain in vivo functionality, such elements have been minimally explored experimentally. In this study, we describe the in-depth characterization of an ancient conserved enhancer, Dc2 located near the dachshund gene, which displays a human-Fugu identity of 84 percent over 424 basepairs (bp). In addition to this large overall conservation, we find that Dc2 is characterized by the presence ofmore » a large block of sequence (144 bp) that is completely identical between human, mouse, chicken, zebrafish and Fugu. Through the testing of reporter vector constructs in transgenic mice, we observed that the 424 bp Dc2 conserved element is necessary and sufficient for brain tissue enhancer activity. In vivo analyses also revealed that the 144 bp 100 percent conserved sequence is necessary, but not sufficient, to replicate Dc2 enhancer function. However, the introduction of two separate 16 bp insertions into the highly conserved enhancer core did not cause any detectable modification of its in vivo activity. Our observations indicate that the 144 bp 100 percent conserved element is tolerant of change at least at the resolution of this transgenic mouse assay and suggest that purifying selection on Dc2 sequence might not be as strong as we predicted or that some unknown property also constrains this highly conserved enhancer sequence.« less

Knowledge of homologies between human and mouse chromosomes is essential for understanding chromosomal evolution and the development of experimental models for human disease. We have reported the identification of a conserved linkage group between human 16p13 and the centromeric portion of the mouse 16. Defining the extent of this linkage conservation has significant biomedical implications since that region of mouse genome contains the Scid mutation and the human 16p13 contains genes that are involved in DNA repair and certain types of human leukemia as well as other diseases such as Rubinstein-Taybi Syndrome. Here, this conserved linkage group has been definedmore » and expanded. It now contains 5 genetic loci and spans more than 3 Mb in human and 23 cM in mouse. The 5 loci are PRM1,2 (protamine 1 and 2), NOP3 (a subclone of D16S237), GSPT1 (a gene involved in the regulation of G1 to S phase transition), MYH11 (a human smooth muscle myosin heavy chain gene) and MRP (multi-drug resistant-associated protein gene). Using a panel of human-rodent hybrids that are informative for different portions of human 16, we have established the following order on human 16p: telomere-NOP3-PRM1,2-GSPT1-(MYH11,MRP)-centromere. The genes were assigned to the mouse chromosome 16 by a mouse-Chinese hamster somatic cell hybrid panel informative for mouse chromosomes. Linkage analysis using backcross mice informative for the Scid mutation indicated the following order and genetic distance (in cM) in mouse: centromere-Nop3-11.7-Prm1-1.4-Gspt1-8.2-(Myh11,Mrp)-1.4-Scid-telomere.« less

Several lines of evidence now suggest that many of the zinc-finger-containing (ZNF) genes in the human genome are arranged in clusters. However, little is known about the structure or function of the clusters or about their conservation throughout evolution. Here, we report the analysis of a conserved ZNF gene cluster located in human chromosome 19q13.2 and mouse chromosome 7. Our results indicate that the human cluster consists of at least 10 related Kruppel-associated box (KRAB)-containing ZNF genes organized in tandem over a distance of 350-450 kb. Two cDNA clones representing genes in the murine cluster have been studied in detail.more » The KRAB A domains of these genes are nearly identical and are highly similar to human 19q13.2-derived KRAB sequences, but DNA-binding ZNF domains and other portions of the genes differ considerably. The two murine genes display distinct expression patterns, but are coexpressed in some adult tissues. These studies pave the way for a systematic analysis of the evolution of structure and function of genes within the numerous clustered ZNF families located on human chromosome 19 and elsewhere in the human and mouse genomes. 32 refs., 7 figs.« less

The mammalian Distal-less (Dlx) clusters (Dlx1-2, Dlx5-6, and Dlx3-7) have a nested expression pattern in developing visceral (branchial) arches. Genetic regulatory mechanisms controlling Dlx spatial expression within the visceral arches have not yet been defined. Here we show that an enhancer in the Dlx3-7 cluster can regulate the visceral arch specific expression pattern of the Dlx3 gene. We have used a 79-kb transgene construct containing the entire Dlx3-7 bigene cluster with a LacZ reporter inserted in frame in the first exon of the Dlx3 gene. Visceral arch expression is absent when a 4-kb element located within the Dlx3-7 intergenic regionmore » is deleted. A 245-bp element (I37-2) whose DNA sequence is highly conserved between human and mouse located within the 4kb-deleted region can drive visceral arch expression when fused to a hsp68-lacZ reporter transgene construct. Reporter expression is detected in 9.5 and 10.5 days postcoitum transgenic embryos in a manner consistent with the endogenous Dlx3 expression pattern in the mesenchyme of the first and second visceral arches. Thus the I37-2 element is both necessary and sufficient for Dlx3 expression. The I37-2 element contains several putative binding sites for several transcription factors including Dlx and other homeodomain proteins within the evolutionarily conserved region. Significantly, the I37-2 element shows a sequence-match including a Dlx binding site to a cis-element in the Dlx5-6 intermediate region designated mI56i [Zerucha, T., Stuhmer, T., Hatch, G., Park, B. K., Long, Q., Yu, G., Gambarotta, A., Schultz, J. R., Rubenstein, J. L. & Ekker, M. (2000) J. Neurosci. 20, 709-721], despite distant phylogenetic relationship between these clusters. Our results provide evidence for a concerted role for DLX auto- and cross-regulation in the establishment of a nested expression pattern for Dlx3-7 and Dlx5-6 clusters within the visceral arches.« less